Reduction of Top2 via RNAi affects gypsy-induced phenotypes.

RNAi screen using dsRNA made from templates generated with primers directed against this gene results in defects in chromosome condensation and chromosome misalignment on the metaphase spindle when assayed in S2 cells. This phenotype can be observed when the screen is performed with or without Cdc27 dsRNA.

Expression is enriched in embryonic gonads.

Mutant males are sterile, showing disruption of chromosome segregation during both meiotic divisions. The chromosomes segregate to one pole only in more than half of telophases I, resulting in many secondary spermatocytes that completely lack chromosomes. Bipolar spindles are formed in these spermatocytes and the spindles undergo the same dynamic transformations as seen in normal meiotic divisions.

Area matching Drosophila Type II topoisomerase gene, Acc. No. X61209.

Amino acid residue Lys 359 is critical for the catalytic activity of Top2.

In a sample of 79 genes with multiple introns, 33 showed significant heterogeneity in G+C content among introns of the same gene and significant positive correspondence between the intron and the third codon position G+C content within genes. These results are consistent with selection adding against preferred codons at the start of genes.

The functional interaction domain for quinolones is mapped and the site of action is shown to overlap with the domain for etoposide, amsacrine and genistein. Results support the hypothesis that many DNA cleavage-enhancing drugs share a common interaction domain on the enzyme.

Several forms of spontaneous DNA damage are capable of acting as endogenous poisons of Top2.

Apurinic sites located within the base of the 5' overhang generated by Top2-mediated DNA scission cause levels of enzyme-associated DNA breaks to increase. Conversely apurinic sites located immediately outside the cleavage overhang inhibits DNA scission. Results indicate apurinic sites have a profound influence on the activity of Top2 enzyme and act as position-specific Top2 positions.

A chromatin-accessibility complex (CHRAC), which uses energy to increase the general accessibility of DNA in chromatin, has been identified. CHRAC can also function during chromatin assembly, using ATP to convert irregular chromatin into a regular array of nucleosomes with even spacing. CHRAC contains 5 subunits, two of which have been identified as the Iswi and Top2 gene products.

barr protein associates with Top2 protein and affects its activity, this suggests barr protein functions in chromatid decatenation during the metaphase-anaphase transition.

Etoposide^.Top2 interactions (rather than etoposide^.DNA interactions) mediate enzyme-DNA cleavage complex formation. Maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than affinity of the drug to site-specific enzyme^.DNA complexes.

Staurosporine inhibits catalytic activity of Top2 by blocking the transfer of phosphodiester bonds from DNA to the active tyrosine site. Other inhibitors most likely inactivate Top2 by alkylation of essential amino acids.

Abasic sites are potent enhancers of double strand DNA cleavage mediated by Top2. Results suggest abasic sites represent cellular Top2 poisons and implies anticancer drugs mimic actions of these endogenous DNA lesions.

Top2 is involved in protein-protein interactions that maintain nuclear localisation of Top2. Top2 has both RNA- and DNA-dependent anchorages in embryo nuclei.

Topoisomerase 2 is distributed throughout interphase nuclei, except for the nucleoli, as judged by antibody staining. In some areas of nuclei localisation is not coincident with chromatin.

The ability of Top2 to cleave an oligonucleotide containing a hairpin has been studied.

Genetic and biochemical properties of a series of insertion linker mutations is used to analyse the domain structure of Top2.

Most Top2 in cell free extracts of 0-2hr embryos appears to be nonnuclear and soluble. This pool of Top2 is detectable in situ and can be detected in extracts from older (16-19hr) embryos and Kc tissue cells. These nonnuclear Top2 containing particles are composed largely of Top2 and an unknown RNA molecule(s).

Top2 is a Z-DNA binding protein, DNA binding characteristics are determined by filter binding and gel retardation. The Z-DNA binding activity of undegraded Top2 may be important in targetting the enzyme both to structural motifs required for chromatin organisation and to sites of local supercoiling.

Anti-Top2 antibodies or epipodophylotoxin VM26 injected into blastoderm embryos inhibits Top2 activity and prevents or hinders anaphase segregation of chromatids. High concentrations of inhibitors block chromosome condensation and the arrangement of chromosomes on the metaphase plate.

The effect of Pkc53E mediated phosphorylation on each step of Top2 catalytic cycle is analysed. As with Casein kinase II (CkIIβ and CkIIβ), stimulation of enzyme activity by Pkc53E correlates with an increased rate of Top2 catalysed ATP hydrolysis. Modification did not affect any step of the enzymes catalytic cycle that preceded the ATPase step.

The effects of several drugs on the DNA strand passage event mediated by Top2 have been analysed.

Top2-mediated DNA cleavage in the 5' region of an Hsp70 gene has been analysed.

Top2 is involved in different functions of the 1731 LTR.

In defining the relationships between drug classes and their interaction domains on Top2 results indicate that there are mechanistic differences between etoposide and a number of other Top2-targeted antineoplastic agents and that the interaction domain of novobocin on the enzyme does not overlap those of several DNA cleavage-enhancing drugs.

The distribution of microinjected fluorescently labelled Top2 has been monitored using time-lapse, three dimensional movies. Experiments show Top2 is localised to the nucleus and on chromosomes is spatially and temporally regulated.

To investigate the potential role for the passage helix in enzyme-mediated DNA cleavage, interactions between Top2 and a 40bp oligonucleotide that contains a specific enzyme recognition/cleavage sequence are characterised. Results indicate that the enzyme has to interact with more than a single double-stranded 40-mer in order for nucleic acid breakage to take place.

The effects of casein kinase II-mediated phosphorylation on the individual steps of the enzymes catalytic cycle are characterised. Results strongly suggest that modification stimulates the activity of Top2 by enhancing the ability of the enzyme to hydrolyse its high energy ATP cofactor.

Top2 can mediate illegitimate recombination events, at least in vitro, by a mechanism that does not rely on double-stranded DNA cleavage or subunit exchange.

The interaction of Top1 and Top2 gene products with transcriptionally active and inactive Act5C, Act57B and Hsp70B has been compared. Topoisomerase II binding to actin and Hsp70B sequences occurs on both transcriptionally active and inactive chromatin. An unusual type of topoisomerase II binding site was identified which is associated with the 5' ends of inactive Hsp70 and actin genes, suggesting that this enzyme may be associated with repression of gene transcription.

Formation of DNA/Top2 complexes have been demonstrated using CsCl equilibrium density centrifugation: three types of complex that are in equilibrium with each other exist. The complex formed in the presence of the VM26 tumour suppressor is extremely stable.

8-methoxycaffeine has been demonstrated to inhibit the activity of Top2 by interfering with the interaction of Top2 with DNA.

Effects of short-wave UV-induced photoproducts on the enzymatic activity of Top2 are investigated. DNA relaxation reaction is inhibited and this correlates with an inhibition of the enzyme DNA strand passage event. Results suggest repair of cyclobutane pyrimidine dimers is important for the catalytic function of Top2.

Top2 enzyme activity has been studied.

EM is used to further characterise the Top2-DNA complex and to directly visualize the position of Top2 molecules on DNA containing sequence-directed bends.

The Top2 gene lies immediately adjacent to the location of the tandem duplication shown to be specific to RanGap^SD chromosomes, and falls within the region from which several RanGap^SD-related transcripts are encoded. However no obvious difference in Top2 transcription could be detected in RanGap^SD mutant flies.

Pre- and post-strand passage DNA cleavage complexes of Top2 serve as physiological targets for structurally disparate antineoplastic drugs.

The effects of CP-67,804 and CP-115,953, two novel quinolone derivatives, on the enzymatic activity of Top2 are characterised. Both drugs enhance the pre- and poststrand passage DNA strand cleavage, neither inhibited the ability to religate cleaved DNA. Results strongly suggest CP-67,804 and CP-115,953 represent a novel class of Top2-targeted drugs.

Top2 mediated cleavage of single-stranded DNA takes pace prior to the addition of SDS in an in vitro system.

Structurally disparate class of Top2 targeted antineoplastic drugs stabilise the enzyme's DNA cleavage complex primarily by interfering with the ability of Top2 to religate DNA.

Top2 enzyme recognises supercoiled DNA at least in part by interacting preferentially with crossover points on DNA.

A number of anon-topo sites have been mapped and sequenced to study the sequence specificity of double strand DNA cleavage by Top2.

DNA molecules with topological knots are detected in a reaction mixture containing circular DNA and purified Top2 enzyme. This knotting reaction has been characterised.

Top2 protein has been purified and characterised.

The relaxation of negatively supercoiled circular DNA by purified Top2 enzyme has been studied.

The cleavage of double strand DNA by Top2 enzyme has been studied.

Top2 is an essential gene that encodes the large subunit of type II DNA topoisomerase, an enzyme believed to play an important role in the condensation, decondensation, and segregation of chromosomes. The enzyme is a major component of the nuclear matrix of Drosophila cells (Berrios, Osheroff and Fisher, 1985) and is distributed along polytene chromosomes paralleling the distribution of the DNA (Heller, Shelton, Dietrich, Elgin and Brutlag, 1986). It is believed to act by passing a DNA segment through a transient double-stranded break in another segment. Major cleavage sites for type II topoisomerase have been found in nontranscribed spacer segments and in the 5' and 3' ends of Hsp70 and the histone genes (Udvardy, Schedl, Sander and Hsieh, 1985). When prepared from embryos, the purified enzyme is made up of a major polypeptide encoded by Top2 of 166,000 daltons, with binding sites for both DNA and ATP, and, in addition, smaller polypeptides of 30,000-40,000 and 132,000-145,000 daltons (Sander and Hsieh, 1983; Shelton, Osheroff and Brutlag, 1983; Heller, Shelton, Dietrich, Elgin and Brutlag, 1986). Protein kinase activity is associated with Drosophila topoisomerase II (Sander, Nolan and Hsieh, 1984; Ackerman, Glover and Osheroff, 1985).